Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Fundamental aspects of mycoplasma cell and molecular biology are poorly understood, despite the significant impact of mycoplasmas on public health and agriculture. Development of more effective means of prevention and control requires a clearer understanding of the basic biological processes of these unique, cell wall-less prokaryotes. M. pneumoniae infections in humans are transmitted by aerosol, followed by the binding of mycoplasmas to the host mucosal blanket and respiratory epithelium. Adherence is mediated largely by a differentiated terminal organelle, a membrane-bound extension of the mycoplasma cell defined by the presence of an electron-dense core. Duplication of the attachment organelle precedes cell division. The focus of this proposal is the organization and assembly of the attachment organelle, with a long-term objective of identifying potential targets for more effective control of M. pneumoniae infections. Aim 1 will examine the structure of the attachment organelle in closer detail, defining the influence of protein HMW2 on the organization of the electron-dense core, examining protein-protein interactions by gel filtration and co-precipitation, and analysing the assembly process using green fluorescent protein fusions. Aim 2 will address the phenotype of a mutant lacking only the adhesin P1. Mycoplasma adherence will be assessed using polarized normal human bronchial epithelial cell monolayers. Recombinant derivatives of P1 will be engineered with epitope tags to establish membrane topography, and functional domains will be characterized through analysis of deletion derivatives. Aim 3 will examine the role of the MPN119 gene product in attachment organelle assembly. This protein shares with other novel cytadherence-associated proteins an acidic proline-rich domain and a domain enriched in aromatic amino acids and glycine, but also has a J-domain, the signature motif of the HSP40 family of molecular chaperones. A non-cytadhering mutant in which MPN119 has been insertionally inactivated by Tn4001 will be characterized in greater detail, including genetic complementation, identification and localization of the gene product, and evaluation of its role in attachment organelle assembly and function. [unreadable] [unreadable] [unreadable]